Aqueous colloidal dispersion for diagnostic tests

ABSTRACT

An aqueous colloidal dispersion for diagnostic or immunodiagnostic tests, comprising non-polymer nuclei surrounded by a hydrophilic copolymer that contains functional groups, a method for the detection of a specifically binding substance or immunochemically active component in a test fluid, and test kit containing the aqueous colloidal dispersion.

This is a continuation of application Ser. No. 07/865,773 filed Apr. 6,1992, now abandoned, which is a file wrapper continuation of U.S. Ser.No. 07/731,373 filed Jul. 16, 1991, now abandoned, which is a filewrapper continuation of Ser. No. 07/434,965 filed Nov. 13, 1989, nowabandoned.

The invention relates to an aqueous suspension for diagnostic orimmunodiagnostic tests, comprising non-polymer nuclei surrounded by ahydrophilic copolymer that contains functional groups, and also to amethod for the preparation of this suspension.

The invention also relates to a method for the detection of aspecifically binding substance or immunochemically active component in atest fluid, and to a reagent and a test kit to be used when employingsaid detection methods.

The abovementioned suspension and a method for preparation of thesuspension are known from U.S. Pat. No. 4,157,323. The particles of thesuspensions described herein are microspheres consisting of a copolymerin which finely divided metal or metal oxide is embedded, as illustratedin FIG. 1.

In immunodiagnostic tests use is frequently made of proteins coupled toa label. Colloidal particles onto which the protein is physicallyadsorbed are then used as label. Disadvantages associated with this are,inter alia, the poor reproducibility of the preparation of the testmaterial and leakage of protein. The meaning of the latter is that theactive protein is not connected in a stable manner to the particles, asa result of which the test sensitivity decreases during storage.

The use of labels to which the proteins can be covalently bonded, asdescribed in U.S. Pat. No. 4,157,323, can be a solution for problems ofthis type. In this case the non-polymer particles constitute the actuallabel. Label is understood to mean the component which can be detectedby reason of a specific property (colour, radioactivity and the like).

The suspension from the abovementioned patent has several disadvantages.Firstly, the solid material in this suspension contains at most 50% byweight of non-polymer nuclei. This signifies a reduction in the testsensitivity and the number of application possibilities compared withlabels with which protein is directly bonded to the nuclei. Secondly, agold sol for the purpose of agglutination tests cannot be used in thissuspension. However, gold particles form a very suitable label by virtueof the characteristic that the colour of a stable gold sol is red whilethe colour changes to blue on flocculating out (agglomeration of theparticles in tests under the influence of the protein to be detected).

When gold nuclei are used in the suspension according to U.S. Pat. No.4,157,323 the colour of the suspended particles is blue or red, but thiscolour cannot change on flocculating out.

When the particles are blue, the gold nuclei are embedded so closely toone another that there can already be considered to be agglomeration ineach particle; see FIG. 2.

When the particles are red, the nuclei are separated from one another bythe copolymer and will remain so even on agglomeration of the polymerspheres, as a result of which the colour does not change (see FIG. 3).

Moreover, this suspension also has a disadvantage in the use of dyestuffsols as nucleus. It is known from EP 0 032 270 that in the finaldetection of dyestuff sols the colour intensity can be intensified byallowing the sol particles to dissolve in an organic solvent. In thecase of dyestuff particles embedded in the thick polymer shell of U.S.Pat. No. 4,157,323 this is, no longer possible.

The solid constituents of a suspension according to the invention have acontent of non-polymer nuclei of at least 50% by weight and thissuspension permits a colour change by agglomeration when gold nuclei areused. Moreover, the polymer which surrounds the nuclei is sufficientlythin to enable--via swelling--dissolution of dyestuff nuclei in anorganic solvent.

The invention consists in that, in a suspension of the abovementionedknown type, the non-polymer nuclei are each separately surrounded bytheir own shell of the copolymer. This is illustrated in FIG. 4.

Particles with this type of construction combine the advantages of thepolymer surface with the characteristics of the nucleus in a ratio whichis as favourable as possible. The content of non-polymer nuclei isdependent on the type of nucleus and the thickness of the polymercasing. In the case of gold this can be more than 90%. The thickness ofthe casing can vary from about 3 to about 70 nm depending, inter alia,on the experimental conditions.

Non-polymer nuclei are for example nuclei of metal, metal oxide, metalcompounds, other inorganic compounds such as silica, organic dyestuffsor organic pigments and emulsion droplets of synthetic, animal,vegetable or mineral oils. Non-polymer nuclei which by virtue of astriking characteristic are best detectable are to be preferred. Theseare gold, by virtue of the already indicated colour change onagglomeration, hematite (Fe₂ O₃) by virtue of the red-brown colour andmagnetite (Fe₃ O₄) by virtue of the magnetic properties.

When dyestuffs are used in colorimetric tests it is possible, with acorrect choice of the dyestuff, to obtain a higher molar absorption--andthus a higher sensitivity--than with metal sols. Moreover--as mentionedabove, an intensification of the colour can be obtained afterwards.

Copolymers which contain functional groups are understood as meaningcopolymers which contain groups such as OH, NH₂, COOH, CHO, SH, NN⁺ Cl⁻,to which proteins can bond directly or, after chemical treatment,covalently.

The invention also relates to a method for the preparation of thesuspension according to the invention described above. With the knownmethod according to U.S. Pat. No. 4,157,323, suspensions are prepared byin situ copolymerization of a mixture of monomers dissolved in water inthe presence of non-polymer particles, the monomer mixture containingthe following types of monomer:

an ethylenically unsaturated monomer which, without hydrolysis or afterhydrolysis, contains at least one covalently bonding functional group;

a hydrophobic monomer;

a linking monomer.

With this method the non-polymer particles are dispersed in the monomersolution. In addition to the fact that the particles formed possess thedisadvantages indicated above, the method also has several drawbacks.Thus, pure polymer spheres without a nucleus are also found to beformed. These are undesired by-products which must be removed. Inaddition, agglomeration of particles is found to be unavoidable. This isprevented by adding a stabilizer, for example a non-ionic surfactant. Inlabels for proteins a substance of this type in general disturbs thebinding and the conformation of the proteins. When particles madeaccording to a method of this type are used for immunodiagnostic testsseparately added surfactants must be removed again. Frequently, however,this is only partly successful.

With the method according to the invention, the aim is to providenon-polymer particles with their own separate copolymer shell, it beingpossible to avoid the use of surfactants and no pure copolymer spheresbeing formed.

The characteristic of the method according to the invention is that astable, colloidal dispersion of the non-polymer particles is used asstarting material and that the monomer mixture is added to this, themonomer mixture being so chosen that the resultant copolymer has acharge of identical sign to that of the original dispersion.

The intended sign of the charge is determined by the electrophoreticmobility. This concept is known to those skilled in the art and requiresno more detailed explanation here.

When the charges of the initial dispersion or emulsion and the shellpolymer are not of identical sign, coagulation of the dispersion takesplace during the formation of the shell in the absence of surfactants.Charges of identical sign are achieved by, during the polymerization,using an initiator which provides charged residual groups with a chargeof the correct sign and/or using charged monomers with a charge of thatsign.

The initial dispersion can be stabilized by peptizing ions, but also byweakly adsorbing surface active substances. Weakly adsorbing surfactantsused in this stage can be removed well by means of microfiltration anddo not hinder the use of the suspension in immunodiagnostic tests.

The ethylenically unsaturated monomers with at least one covalentlybonding functional group can be chosen from monomers with an OH, NH₂,COOH, CHO, SH or NN⁺ Cl⁻ group. Examples of these are ethyleneimine,2,3-dihydroxypropyl methacrylate, maleic acid, acrylamide,methylolacrylamide, (meth)acrylic acid, aldonic acid, allylamides suchas arabinon allylamide, glucon allylamide, α-glucohepton allylamide,lactobion allylamide, sodium vinylsulphonate, methallyl sulphonate,dimethyl aminoethyl methacrylate, vinylpyridine salts, (meth)acrylicacid esters of polyethylene glycol and vinyl-pyridine at low pH.

It is also possible to use monomers which after hydrolysis contain acovalently bonding functional group. Exampled of these are:

vinyl acetate (hydrolysis product: vinyl alcohol);

N-vinyl-tertiary butyl carbamate (hydrolysis product: vinylamine);

glycidyl methacrylate (hydrolysis product: 2,3-dihydroxypropylmethacrylate);

diethyl maleate (hydrolysis product: maleic acid).

The hydrophobic monomers can be chosen from monomers with a solubilityin water of at least 35 g/l at 20° C. Examples of these are styrene,butadiene, butyl acrylate, vinylidene chloride, vinyl chloride, ethene,methyl methacrylate, ethyl acrylate, vinyl esters, diethyl maleate,glycidyl methacrylate and 2,3-epithiopropyl methacrylate.

The most advantageous hydrophobic monomers are, however, those whichpossess a hydrolysable group, so that a hydrophilic unit is formed inthe polymer. Examples of these are the same hydrolysable monomers asdescribed above.

Examples of linking monomers are N,N-methylenebisacrylamide, ethyleneglycol dimethacrylate, diallyl phthalate, pentaerythritol triacrylateand N,N-diallyltartaric acid diamide. When monomers such as glycidylmethacrylate and methylol acrylamide which are already linkable arechosen as the hydrophilic and hydrophobic monomer, the addition of aseparate linking monomer is superfluous. A linking agent is also notnecessary when the copolymer is sufficiently insoluble in thepolymerization medium. This is the case with a copolymer of styrene andacrylamide.

The ratio in which the monomers can be chosen is dependent on whichmonomers are chosen.

It is essential that the shell polymer formed possesses stabilizingproperties. This can be demonstrated by allowing the monomer mixture topolymerize in the absence of the nucleus particles, while surfactantsmay also not be present. A copolymer which forms a stable latex with aparticle size of between 50 and 300 nm now forms from suitable monomermixtures.

A reagent or an immunochemical reagent also belongs to the invention.The term reagent signifies that the hydrophilic copolymer, whichsurrounds the non-polymer nucleus, is provided with a reactant.

Reactants which can be used are substances with which either a receptoror a ligand in a receptor-ligand combination can react. In suchreceptor-ligand combinations receptor and ligand have a direct orindirect bonding affinity for one another. Suitable receptor-ligandcombinations are, for example, avidine-biotine or a DNA-DNA or DNA-RNAhybrids. The said reagent can then be used in a method for the detectionof a specifically binding substance in a test fluid, this substancehaving a bonding affinity for the reactant present in the reagent.

The term immunochemical reagent signifies that the hydrophiliccopolymer, which surrounds the non-polymer nucleus, is provided with animmunochemically active substance (as reactant). An antibody, an antigenor hapten can be used as immunochemically active substance.

This immunochemical reagent can then be used in a method for thedetection of immunochemical active components in a test fluid. Theimmunochemical reaction which should take place when the detectionmethod is used is preferably a sandwich reaction, an agglutinationreaction, a competition reaction or an inhibition reaction.

In order, for example, to demonstrate an antigen in a test fluid, anantibody directed against the antigen can be bound to a suitablesupport, after which the test fluid is brought into contact with thesupport and the presence of immune complexes, formed between the antigenin the test fluid and the antibody, is detected by adding the suitableimmunochemical reagent according to the invention to the support afterthe immune complex has formed.

Supports which can be used are, the inner wall of a microtest well, atube or capillary, a membrane, filter, test strip or the surface of aparticle, such as, for example, a latex particle, an erythrocyte, adyestuff sol, a metal sol or metal compound as sol particle.

A test kit according to the invention must contain, as essentialconstituent, said reagent or immunochemical reagent.

The invention will be illustrated below with the aid of the followingnon-limiting examples and FIGS. 1 to 4 inclusive.

FIG. 1 shows the particles of a suspension according to the prior art.Here various nucleus particles are embedded per particle.

FIG. 2 shows particles of a suspension according to the prior art inwhich the nuclei are embedded close to one another.

FIG. 3 shows particles of a suspension according to the prior art inwhich the nuclei are separated from one another by the copolymer.

FIG. 4 shows the particles of a suspension according to the invention.In this case each nucleus possesses its own copolymer shell.

EXAMPLE 1

Coating of gold particles with a polymer shell

A gold seed sol with a particle size of about 20 nm is prepared by themethod of Frens (Nature, Physical Sci. 241 (1973), 20). The gold sol hasa solids content of 0.006% by weight. 80 ml of this sol are warmed to70° C. in a double-walled, thermostat-controlled reactor at a stirringspeed of 200 revolutions per minute. 0.1 g potassium persulphate and0.06 g sodium bicarbonate dissolved in 5 ml distilled water are thenadded to the gold sol, followed by 7 ml of the following monomersolution:

2 g methyl methacrylate

1 g sodium vinylsulphonate

1 g N-methylenebisacrylamide

50 ml distilled water

50 ml methanol

The mixture is stirred at 70° C. for 18 hours and then cooled to roomtemperature. The centrifuging tests show that no separate polymerparticles have formed. Visible light absorption spectra show that noclusters of particles are present and that a polymer shell has formed.Dynamic light scattering tests and transmission electron microscopy showthat the shell thickness is 77 nm.

EXAMPLE 2

Monomer mixture with hydrolysable hydrophobic monomer

2.1 Coating of gold particles with a thin polymer shell

Seed sol preparation

A gold sol is prepared by reduction of a tetrachloroauric acid solutionwith sodium citrate according to the method described by Frens (Nature,Physical Sci. 241 (1973), 20). The solids content is 0.032% by weightgold. The mean particle size is 55 nm. The sol prepared in this way iscolloidally stable without the addition of surfactants.

Coating procedure

800 ml of the seed sol are transferred to a thermostat-controlled1-liter reactor and warmed to 70° C. with slow stirring (200 revolutionsper minute, anchor stirrer). 10 ml of a solution of 0.32 g potassiumpersulphate and 0.2 g sodium bicarbonate in 50 ml distilled, deionizedwater are then added dropwise to the gold sol. 25 ml of a solution withthe following composition is then added dropwise to the stirred, warmgold sol in the course of 1 hour:

Composition of monomer feed

0.75 g glycidyl methacrylate

0.38 g sodium vinylsulphonate

0.38 g N-methylenebisacrylamide

50 ml distilled water

50 ml methanol

During the same period the remaining portion of the initiator/buffersolution is added. After adding the monomers and initiator, the mixtureis stirred for a further 15 hours at 70° C. When the same experiment iscarried out with water in place of the volume of gold sol a stable latexwith a particle size of approximately 100 nm forms.

Characteristics of the coated gold sol

200 ml of the sol prepared in this way are purified by microfiltrationwith 6,000 ml distilled water. Analyses by transmission electronmicroscopy and quasi-elastic light scattering show that each individualgold particle is coated with an 11 nm-thick polymer shell. Less than 5%polymer particles without gold nucleus are formed. Each coated particlecontains a single gold nucleus.

The purified, micro-filtered sol remains colloidally stable in a 0.2Msodium chloride solution in water, even after 60 hours. In contrast, thestarting seed sol flocculates with a sodium chloride concentration of0.04M.

The colour of the coated sol is virtually identical to that of the seedsol. Maximum absorption occurs at wavelengths of 539 and 533 nm. Onflocculation, for example under the influence of sodium chloride, thecharacteristic colour change from red-pink to blue and finallygrey-colourless is detected for both the seed sol and the coated sol.

2.2 Chemical, covalent bonding of immunoglobulin G (anti-human choriongonadotrophin, a-hCG) to a polymer-coated gold sol

Introduction of aldehyde groups

100 ml of the encapsulated, micro-filtered gold sol from Example 2.1 aremixed with 8.7 ml of a 0.5M sodium periodate solution at pH=4.6.

The coated, micro-filtered gold sol mixed with 8.7 ml distilled water istaken as a blank experiment.

These mixtures are stored for 75 min at room temperature. The oxidationis then stopped by the addition of 304 ml ethylene glycol, after whichthe mixtures are stirred for a further 60 min. The sols are thenmicro-filtered with the 20-fold volume of distilled water.

Chemical binding of a-hCG 293A to the coated gold sol provided withaldehyde groups

100 ml of the gold sol provided with aldehyde functional groups aremixed with 5 ml of the buffered a-hCG solution (borate buffer,pH=9^(a)). For comparison, the control (blank) is treated in the sameway.

The mixtures are incubated at room temperature for 18 hours and thenfiltered through a coarse nylon filter.

These sols are then washed twice with tris buffer, pH=8^(b)) bycentrifuging the sols for 60 min at 700 g.

The sediments are resuspended in tris buffer.

The coated gold sols which have been subjected to this treatment aretested for their immunological activity using a Predictor stick (ChefaroInternational). For this purpose 0.3 ml of conjugate (opticaldensity=8.33) is mixed with 0.2 ml urine, containing 0, 50 and 1,000International Units (I.U.) hCG/l respectively. A stick coated withmonoclonal a-hCG (147B) is placed in the gold sol/urine mixture andincubated for 30 minutes at room temperature. The stick is then washedwith water and the colour read off. The results are given below in TableI.

                  TABLE I                                                         ______________________________________                                        Concentration hCG                                                                              Control  Aldehyde                                            I.U./l           sol      sol                                                 ______________________________________                                        0                -        -                                                   50               -        +                                                   1,000            -        +                                                   ______________________________________                                         - signifies no coloration visible on the stick                                + redpink coloration visible on the stick                                

a) borate buffer composition:

solution A: 0.2M boric acid+0.2M potassium chloride

solution B: 0.2M sodium carbonate solution A is brought to pH=9.0 byadding solution B.

Buffered a-hCG solution: 1 part by volume a-hCG (9.9 mg ml⁻¹) togetherwith 8.4 parts by volume of the 0.2M borate buffer.

b) tris buffer composition:

0.25M tris(2-amino-2-(hydroxymethyl)-1,3-propanediol

0.25M sodium chloride

1.29 g bovine serum albumin/l

0.025 g thiomersal/l brought to pH=8 by adding concentrated hydrochloricacid.

2.3 Preparation of a thick polymer shell around individual acidparticles

Coating

The same procedure as described in Example 2.1 is chosen for coating thegold particles, except for the composition of the monomer feed.

1.70 g glycidyl methacrylate

0.85 g sodium vinylsulphonate

0.85 g N-methylenebisacrylamide

50 ml distilled water

50 ml methanol

200 ml of the sol thus obtained are micro-filtered with 6,000 mldistilled water and characterized. Using transmission electronmicroscopy a polymer shell thickness of a good 25 nm is estimated; fromquasi-elastic light scattering a shell thickness of 40 nm is derived,which is an indication of the swelling of the polymer shell in water.The majority of the particles (>95%) are made up of a single goldnucleus surrounded by a polymer shell.

The coated, micro-filtered gold sol does not flocculate in 0.4M sodiumchloride solution, even after 60 hours. Flocculation does occur atsignificantly higher sodium chloride concentrations, but the colourchange, characterizing for the flocculation of uncoated gold sols, thenno longer takes place. Instead, red-pink flocs are detected. If the seedgold sol is replaced by the corresponding volume of water, thepolymerization of the monomers yields a stable latex with a particlesize of a good 120 nm.

EXAMPLE 3

Coating of a dyestuff sol

3.1 Seed sol preparation

50 gram Palanil light red (a disperse dyestuff slurry BASF No. 7764060)are stirred in 1,000 ml distilled water for 45 minutes at roomtemperature. This dyestuff sol is purified by six successive washes withdistilled water. The first 5 wash steps are carried out by centrifugingat an acceleration of 2,000 g for 30 min, followed by redispersion indistilled water. The final centrifuging step is carried out at anacceleration of 125 g for 60 min. This purification procedure iseffective in removing surplus surfactant. Moreover, some fractionationoccurs. The crude sol has a surface tension of 43.5 dyne/cm; thepurified sol has a surface tension of 71.1 dyne/cm, both measured at asolids content of 4.8 g dyestuff/l.

3.2 Coating

125 ml dilute, washed dyewstuff sol (0.145%) are warmed to 70° C. andstirred (200 rotations per minute) in a reactor. 12.5 ml of a solutionof 0.10 g sodium bicarbonate and 0.10 g potassium persulphate in waterare then added. 12.5 ml of a monomer solution of the followingcomposition is then metered in with a peristaltic pump in the course of1 hour:

monomer feed

2 g glycidyl methacrylate

1 g sodium vinylsulphonate

0.5 g N-methylenebisacrylamide

50 ml distilled water

50 ml methanol

The composite sol is cooled after 16 hours and 100 ml were purified bymicrofiltration with 2,500 ml distilled water. The diameter of thecoated sol is 86 nm larger than that of the initial sol, which is 316nm. Centrifuging the composite sol in a 52 weight/weight % glucose/watersolution with a density of 1.23 g/ml results in red particles sedimentedon the bottom of the tube. No separate polymer particles are detected onthe top of the supernatant liquor. With "sedimentation field flowfractionation" experiments also no separate polymer particles aredetected.

The effect of the surface modification on the adsorption of proteins issubstantial: two 4-ml tubes are filled with 1 ml uncoated, purified soland 1 ml coated, purified dyestuff sol, respectively. Each tube contains0.054 g dyestuff. 1.75 ml phosphate buffer*) of pH=7.4 and then 0.75 mlof a solution containing 2.67 mg/ml bovine serum albumin (R-type,Organon Teknika) are added to these tubes.

The tubes are shaken at room temperature for 2 hours and thencentrifuged. The bovine serum albumin content of the supernatant liquoris determined by HPSEC (high performance size exclusion chromatography).0.28 mg/m² protein has been adsorbed on the uncoated, purified sol; noprotein adsorption is detected on the coated sol.

3.3 Chemical, covalent bonding of immunoglobulin G (anti-human choriongonadotrophin, a-hCG) to a polymer-coated dyestuff sol

3.3.1. Introduction of aldehyde groups.

The encapsulated, micro-filtered dyestuff sol from Example 3.2 and othersamples with a different layer thickness in respect of the polymercoating are provided with aldehyde groups as described under 2.2.

3.3.2. Chemical bonding of a-hCG 293A to the coated dyestuff solprovided with aldehyde groups.

The coupling of a-hCG to the sols provided with aldehyde functionalgroups (see 3.3.1.) is carried out as described under 2.2.

3.4 Immunoassay.

The (a-hCG)-dyestuff sol conjugates made in this way are tested fortheir immunological activity in a sandwich immunoassay for hCG, aslikewise described under 2.2, by incubating a a-hCG (147B) coateddipstick (Predictor stick; Chefaro International) at room temperature ina mixture of hCG-containing urine and conjugate ##EQU1## The stick isthen rinsed with water and the colour is read off. The results are givenin Table II.

                                      TABLE II                                    __________________________________________________________________________    Dyestuff sol-(a-hCG) conjugates tested in a dipstick sandwich                 immunoassay for hCG (Predictor Stick, Chefaro International).                 Concentration hCG                                                                       Incubation                                                          (I.U./l)  time (hours)                                                                         ACI-113*                                                                           ACI-124*                                                                           ACI-125*                                                                           ACI-126*                                                                           ACI-127*                                 __________________________________________________________________________    0         0.5    -    -    -    -    -                                        200       0.5    +/-  +/-  -    -    -                                        1,000     0.5    +    +    +/-  -    -                                        10,000    0.5    +    +    +    +/-  +/-                                      0         1.0    -    -    -    -    -                                        200       1.0    +/-  +/-  +/-  -    -                                        1,000     1.0    +    +    +    +/-  -                                        10,000    1.0    +    +    +    +    +                                        0         2.0    -    -    -    -    -                                        200       2.0    +/-  +    +/-  -    -                                        1,000     2.0    +    +    +    +/-  +/-                                      10,000    2.0    +    +    +    +    +                                        __________________________________________________________________________     *.sup.) conjugate based on polymercoated dyestuff sols with the following     variable layer thickness (measured with the aid of dynamic light              scattering/QELS):                                                             ACI113 : 86 nm                                                                ACI124 : 88 nm                                                                ACI125 : 58 nm                                                                ACI126 : 30 nm                                                                ACI127 : 35 nm                                                                + redpink colour visible on white stick                                       - no visible coloration of the white stick                               

EXAMPLE 4

The coating of gold particles above the solubility limit of the shellpolymer

800 ml of the seed gold sol (0.032% by weight; see Example 1 for thepreparation of this seed sol) are warmed to 70° C. in athermostat-controlled reactor with reflux condenser and stirrer. 10 mlof a solution of 0.32 g potassium persulphate and 0.32 g sodiumcarbonate in 50 ml distilled water are added dropwise and 10 ml of amonomer solution of the following composition is added dropwise in thecourse of 1 hour:

monomer feed

12 g vinyl acetate

4 g diethyl maleate

3 g diethyltartaric acid diamide

50 ml water

50 ml methanol

The remainder of the initiator/buffer solution is added during theaddition of the monomers. 24 hours after the addition of the monomersthe reactor is cooled to room temperature and the sol is purified bymicrofiltration with a 25-fold volume of water relative to the originalsol volume. No polymer shell can be detected around the particles andthe colloidal stability is just as low as that of the "uncoated" seedsol. Repetition of the polymerization procedure using water in place ofthe seed sol does not yield an insoluble polymer: the solution remainsclear. However, if the polymerization procedure is carried out by addingnot 10 ml but 80 ml of the above monomer solution in the course of 1hour a latex is obtained with a particle size of 152 nm in the absenceof seed sol. In the presence of gold particles a polymer shell is formedaround the gold particles with a thickness of 27 nm. The colloidalstability is also distinctly greater than that of the seed sol.

EXAMPLE 5

Coating of gold particles below the solubility limit of the coatingmonomer

100 ml of a 0.032% by weight seed gold sol (for preparation seeExample 1) are warmed to 70° C. under a blanket of nitrogen. 5 ml of asolution of 0.1 g potassium persulphate and 0.1 g sodium bicarbonate areadded dropwise to this sol. 2.2 ml of a monomer solution with thefollowing composition:

monomer feed composition

5 g styrene

1 g acrylamide

80 ml methanol

is then added with the aid of a peristaltic pump.

After stirring and warming to 70° C. for 24 hours, the reactor is cooledto room temperature. The gold sol is micro-filtered through a 25-foldvolume of water. The purified sol proves able to withstand 0.10M sodiumchloride after standing for 24 hours. No second generation of polymerparticles has formed. Under these conditions styrene monomer dissolvescompletely in the reaction mixture.

When this polymerization is carried out without gold sol but with waterin its place, a colloidal stable latex with a particle size of 120 nm isformed.

However, if a higher concentration of feed monomer is used by adding 3.4ml of the following composition:

monomer feed

10 g styrene

2 g acrylamide

80 ml methanol

this yields a new generation of polymer particles. At this highermonomer concentration styrene monomer no longer dissolves in themixture. Moreover, the gold particles treated in this way prove littleable to withstand salt and after purification the sol alreadyflocculates with 0.04M NaCl, the same concentration as the starting sol(seed sol). It can be concluded from this that in this case no polymercoating of the particles has taken place. In contrast to the goldparticles, the newly formed polymer particles are able to withstandsodium chloride concentrations higher than 0.1M.

EXAMPLE 6

6.1 Comparison example analogous to U.S. Pat. No. 4,157,323 Example 3

1.8 g hydroxyethyl methacrylate

0.3 g N-methylenebisacrylamide

0.6 g acrylamide

0.3 g methacrylic acid

are dissolved in 90 ml deionized (Milli-Q) water in a double-walledreactor.

The solution is warmed to 70° C. and stirred at 200 rotations perminute. 0.1 g potassium persulphate and 0.1 g sodium bicarbonatedissolved in 10 ml water are then added.

After about 3-5 min large white flocs form. This indicates the formationof non-colloidal polymer.

6.2 Comparison example analogous to U.S. Pat. No. 4,157,323 Example 11

0.64 g hydroxyethyl methacrylate

0.70 g methyl methacrylate

0.64 g methacrylic acid

0.224 g ethylene glycol dimethacrylate

are dissolved in 90 ml deionized (Milli-Q) water in a double-walledreactor.

Room temperature is chosen as the reaction temperature. 0.08 g potassiumpersulphate and 0.04 g sodium bisulphite dissolved in 10 ml of water areadded to the mixture.

After 15 min an unstable latex (flocculated polymer) is detected.

I claim:
 1. An aqueous colloidal dispersion for diagnostic testscontaining particles comprising non-polymer nuclei surrounded by ahydrophilic copolymer that contains functional groups, wherein eachnon-polymer nucleus makes up at least 50% by weight of each particle andeach nucleus is surrounded by its own shell of said hydrophiliccopolymer, and wherein said non-polymer nuclei function as detectablecolorimetric labels and are selected from the group consisting of goldsols and dyestuff sols.
 2. The aqueous colloidal dispersion according toclaim 1, wherein the diagnostic test is an immunodiagnostic test.
 3. Theaqueous colloidal dispersion according to claim 1, wherein saidhydrophilic copolymer further comprises a reactant selected from thegroup consisting of ligands and receptors bound to said functionalgroups.
 4. The aqueous colloidal dispersion according to claim 2,wherein said hydrophilic copolymer further comprises an immunochemicallyactive substance bound to said functional groups.
 5. A method for thedetection of a specifically binding substance in a test fluid,comprising combining the aqueous colloidal dispersion according to claim3 with the test fluid, said specifically binding substance havingbinding affinity for the reactant present on the particles of thecolloidal dispersion, and detecting conjugates comprising thespecifically binding substance and particles of the colloidal dispersionto detect said specifically binding substance.
 6. A method for thedetection of an immunochemically active component in a test fluid,comprising combining the aqueous colloidal dispersion according to claim4 with the test fluid, said immunochemically active component havingbinding affinity for the immunochemically active substance present onthe particles of the colloidal dispersion, and detecting conjugatescomprising the immunochemically active component and particles of thecolloidal dispersion to detect said immunochemically active component.7. A test kit to be used in a diagnostic test comprising the aqueouscolloidal dispersion according to claim
 3. 8. A test kit to be used inan immunoassay comprising the aqueous colloidal dispersion according toclaim 4.